An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation.

Agarraberes FA, Terlecky SR, Dice JF - J. Cell Biol. (1997)

Bottom Line:
The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected.The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3.These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

Affiliation: Sackler School of Graduate Biomedical Sciences, Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACTPrevious studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules. Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

Mentions:
Previous studies have shown that mAb 13D3 recognizes hsc73 but not the major heat-inducible hsp70 of human fibroblasts in immunoblots (Terlecky et al., 1992). mAb 13D3 also recognized hsc73, or another as yet unidentified hsp70, in a cellular membrane fraction (see Materials and Methods; Fig. 1 A, lane 1) but did not recognize what we presume to be grp78 (Fig. 1 A, lane 2, upper band). Both hsc73 and grp78 could be visualized by mAb 7.10, which is known to recognize several members of the hsp70 family (Kurtz et al., 1986; Fig. 1 A, lane 2). In addition, mAb 13D3 did not recognize the yeast hsp70, SSA1p (Fig. 1 B, lane 1), which is recognized by mAb 7.10 (Fig. 1 B, lane 2).

Mentions:
Previous studies have shown that mAb 13D3 recognizes hsc73 but not the major heat-inducible hsp70 of human fibroblasts in immunoblots (Terlecky et al., 1992). mAb 13D3 also recognized hsc73, or another as yet unidentified hsp70, in a cellular membrane fraction (see Materials and Methods; Fig. 1 A, lane 1) but did not recognize what we presume to be grp78 (Fig. 1 A, lane 2, upper band). Both hsc73 and grp78 could be visualized by mAb 7.10, which is known to recognize several members of the hsp70 family (Kurtz et al., 1986; Fig. 1 A, lane 2). In addition, mAb 13D3 did not recognize the yeast hsp70, SSA1p (Fig. 1 B, lane 1), which is recognized by mAb 7.10 (Fig. 1 B, lane 2).

Bottom Line:
The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected.The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3.These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

Affiliation:
Sackler School of Graduate Biomedical Sciences, Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACTPrevious studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules. Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.